BMC Bioinformatics BioMed Central Research article Open Access Components of the antigen processing and presentation pathway revealed by gene expression microarray analysis following B cell antigen receptor (BCR) stimulation Jamie A Lee†1, Robert S Sinkovits†3, Dennis Mock†3, Eva L Rab1, Jennifer Cai1, Peng Yang1, Brian Saunders3, Robert C Hsueh2, Sangdun Choi5, Shankar Subramaniam3,4, Richard H Scheuermann*1,3 and in collaboration with the Alliance for Cellular Signaling Address: 1Department of Pathology, Laboratory of Molecular Pathology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA, 2Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA, 3San Diego Supercomputer Center, University of California, San Diego, California 92122, USA, 4Department of Bioengineering, University of California, San Diego, California 92122, USA and 5Division of Biology, California Institute of Technology, Pasadena, CA, USA Email: Jamie A Lee - [email protected]; Robert S Sinkovits - [email protected]; Dennis Mock - [email protected]; Eva L Rab - [email protected]; Jennifer Cai - [email protected]; Peng Yang - [email protected]; Brian Saunders - [email protected]; Robert C Hsueh - [email protected]; Sangdun Choi - [email protected]; Shankar Subramaniam - [email protected]; Richard H Scheuermann* - [email protected]; in collaboration with the Alliance for Cellular Signaling - [email protected] * Corresponding author †Equal contributors Published: 02 May 2006 Received: 21 September 2005 Accepted: 02 May 2006 BMC Bioinformatics 2006, 7:237 doi:10.1186/1471-2105-7-237 This article is available from: http://www.biomedcentral.com/1471-2105/7/237 © 2006 Lee et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Activation of naïve B lymphocytes by extracellular ligands, e.g. antigen, lipopolysaccharide (LPS) and CD40 ligand, induces a combination of common and ligand-specific phenotypic changes through complex signal transduction pathways. For example, although all three of these ligands induce proliferation, only stimulation through the B cell antigen receptor (BCR) induces apoptosis in resting splenic B cells. In order to define the common and unique biological responses to ligand stimulation, we compared the gene expression changes induced in normal primary B cells by a panel of ligands using cDNA microarrays and a statistical approach, CLASSIFI (Cluster Assignment for Biological Inference), which identifies significant co-clustering of genes with similar Gene Ontology™ annotation. Results: CLASSIFI analysis revealed an overrepresentation of genes involved in ion and vesicle transport, including multiple components of the proton pump, in the BCR-specific gene cluster, suggesting that activation of antigen processing and presentation pathways is a major biological response to antigen receptor stimulation. Proton pump components that were not included in the initial microarray data set were also upregulated in response to BCR stimulation in follow up experiments. MHC Class II expression was found to be maintained specifically in response to BCR stimulation. Furthermore, ligand-specific internalization of the BCR, a first step in B cell antigen processing and presentation, was demonstrated. Conclusion: These observations provide experimental validation of the computational approach implemented in CLASSIFI, demonstrating that CLASSIFI-based gene expression cluster analysis is an effective data mining tool to identify biological processes that correlate with the experimental conditional variables. Furthermore, this analysis has identified at least thirty-eight candidate components of the B cell antigen processing and presentation pathway and sets the stage for future studies focused on a better understanding of the components involved in and unique to B cell antigen processing and presentation. Page 1 of 19 (page number not for citation purposes) BMC Bioinformatics 2006, 7:237 http://www.biomedcentral.com/1471-2105/7/237 Background microarray analysis with bioinformatics analyses and Naïve mature B cells in peripheral lymphoid organs functional assays, we have identified a set of specific genes respond to a variety of extracellular signals through the that appear to be involved in BCR-mediated antigen cap- activation of signal transduction pathways initiated by the ture, vesicle function and vesicle trafficking during B cell B cell antigen, pattern-recognition, cytokine and chemok- antigen processing and presentation. These data provide a ine receptors. B cell responses to signaling depend on the foundation for the molecular characterization of this combination of ligands present, and include activation, important immunological process. proliferation, migration, differentiation, isotype class switching, somatic hypermutation, anergy, and apoptosis Results [1,2]. Once activated, B cells can also serve as antigen pre- Microarray analysis of ligand-treated B lymphocytes senting cells that preferentially present antigens recog- Purified B lymphocytes were treated in culture with 32 dif- nized by their specific BCR. In contrast, dendritic cells and ferent ligands over a timecourse of 30 min, 1 hr, 2 hr, and macrophages present varied antigens that are acquired less 4 hr. A detailed description of the data set has been pub- specifically through phagocytosis, macropinocytosis and lished [8]. Following filtering, normalization and SAM receptor-mediated endocytosis via pattern-recognition analysis, genes that were differentially expressed by each receptors such as the mannose receptor. ligand in comparison with time-matched, untreated con- trols were identified. Of the 32 ligands, CD40L, LPS, and BCR-specific antigen processing and presentation is initi- AIG caused the most gene expression changes, especially ated by BCR-mediated signal transduction triggered by at the 4 hr timepoint (Figure 1B). Further analysis focuses antigenic stimulation [3,4]. Antigen is then internalized on these three immunologically-important ligands. Cate- by receptor-mediated endocytosis and trafficked through gorical values of 1, -1, and 0 (representing significantly endosomes for acidification and fusion with lysosomes upregulated, downregulated, or unchanged) were used to containing pH-sensitive hydrolytic enzymes for antigen group genes together based on their expression response processing. Endolysosomes containing processed anti- patterns (Table 1). Genes not differentially expressed genic peptides fuse with Golgi-derived vesicles containing under at least one treatment condition were omitted from MHC class II molecules assembled with invariant chain further analysis. Based on 3 ligand combinations and a (Ii). The CLIP fragment of Ii bound in the cleft of the class possibility of 3 outcomes for each ligand (1, -1, 0), there II aβ dimer is replaced by antigen-derived peptides and are a possible 33 or 27 gene clusters, with the (0, 0, 0) out- the complex trafficked to the cell surface through vesicle come excluded since only genes that were differentially secretory pathways. expressed under at least one treatment condition were selected (26 possible gene clusters). In our dataset, we It is well known that B cell antigen processing and presen- only observe 19 of these possible 26. A variety of different tation mediated through the BCR far exceeds the effi- expression patterns were observed. For example, Gene ciency of presentation of the same antigen by Cluster #1 contains genes that are upregulated by all three macrophages or dendritic cells [5]. The mechanism giving ligands, whereas Gene Cluster #14 contains genes that are rise to this increased efficiency has not been fully deter- only upregulated in response to LPS. mined but appears to be a unique aspect of BCR-mediated antigen capture and processing as opposed to changes in CLASSIFI analysis links gene clusters to cellular physiology the basic antigen processing and presentation machinery Microarray expression data can be full of experimental [6]. One mechanism that may contribute to efficiency is and biological noise, and many microarray probes are not accelerated trafficking of BCR/antigen complexes to Class well characterized. We developed a gene cluster classifica- II containing vesicles inside the cell [7]. However, the tion method that circumvents these limitations and links molecular mediators of this vesicle trafficking, especially biological function with gene expression patterns derived those components uniquely involved in the highly effi- from microarray experiments. This method, termed CLAS- cient B cell antigen processing and presentation pathway, SIFI, uses Gene Ontology™ annotation to identify signifi- have remained largely unknown. cant co-clustering of genes with similar biological properties, based on the postulate that genes involved in We analyzed a B cell microarray dataset comparing the the same biological process would be coordinately responses of normal splenic B cells to 32 individual lig- expressed. ands. This study was designed to determine functionally important input signals to contribute to the understand- CLASSIFI utilizes the gene description database developed ing of normal B cell